Abstract: Multi-tissue fatty acid profile analysis reflects dietary information of organisms at different time scales, which plays an important indicative role in exploring trophic niche partitioning among large apex predators. In this study, we investigated the trophic niche partitioning of four shark species in the upper middle layer of tropical Atlantic Ocean (Prionace glauca, Isurus paucus, Pseudocarcharias kamoharai, I. oxyrinchus) by measuring the fatty acid profiles of their muscle and liver, to reveal the short-term and long-term feeding strategies and nutritional relationships. The results show that the four shark species had diverse degrees of dietary or habitat variation, and high trophic niche overlap was found between I. paucus and P. kamoharai, with intense dietary and habitat competition. However, there was trophic niche partitioning between I. paucus and I. oxyrinchus, indicating their dietary separation. P. glauca had relatively longer trophic niche width, indicating greater environmental plasticity, while the smaller one of P. kamoharai indicates dietary specialization. Comparison of liver and muscle fatty acid profiles reveals variable resource allocation patterns among sharks over time, with the highest overlap of trophic niche between the muscle of I. oxyrinchus and P. glauca, but separation of livers suggests intense competition for long-term feeding. The results help to understand the coexistence mechanisms of pelagic sharks.